Process for the preparation of aβ-lactam antibiotic

ABSTRACT

Process for the preparation of a β-lactam antibiotic in which a β-lactam nucleus is subjected to an enzymatic acylation reaction with the aid of an acylation agent at a molar ratio of acylation agent/β-lactam nucleus of less than 2.5, with the acylation agent and/or the β-lactam nucleus being supersaturated in the reaction mixture during at least part of the acylation reaction. In the process, a concentrated slurry or solution, for instance, of the β-lactam nucleus and/or the acylation agent with a different pH or a higher temperature than the pH or temperature at which the acylation reaction is carried out is added to the reaction mixture during the acylation reaction. Both the β-lactam nucleus and the acylation agent may be supersaturated in the reaction mixture.

This application is a 371 of PCT/NL98/00570 filed Oct. 1, 1998.

The invention relates to a process for the preparation of a β-lactamantibiotic in which a β-lactam nucleus is subjected to an enzymaticacylation reaction with the aid of an acylation agent at a molar ratioof acylation agent/β-lactam nucleus of less than 2.5.

A similar process is disclosed in for example WO-A-96/23897.

The yield of β-lactam antibiotic to be achieved in the enzymaticacylation reaction according to the prior art per amount of β-lactamnucleus employed and per amount of acylation agent employed is ingeneral relatively low inasmuch as the β-lactam nuclei and/or theβ-lactam antibiotics often are relatively instable, whilst the reactiontime is relatively long because of the usually low solubility of thereactants. Moreover, at the relatively low acylation agent to β-lactamnucleus ratio mentioned, only a relatively low yield of β-lactamantibiotic can be achieved per amount of β-lactam nucleus employed.

The invention provides a process in which in an enzymatic acylationreaction a shorter reaction time is achieved and a higher yield ofβ-lactam antibiotic is achieved per amount of β-lactam nucleus and/orper amount of acylation agent employed, with the ratio of the amount ofacylation agent employed to β-lactam nucleus being relatively low.

This is achieved according to the invention in that the acylation agentand/or the β-lactam nucleus are/is supersaturated in the reactionmixture during at least part of the acylation reaction.

The applicant has found that it is possible to achieve a high degree ofsupersaturation of the β-lactam nucleus and/or the acylation agent inthe reaction mixture and that, especially surprisingly, thesupersaturation can be kept stable for hours. This allows theconcentration of the β-lactam nucleus and/or the dissolved acylationagent to be strongly increased, so that the reaction proceeds morerapidly, with less degradation of the β-lactam antibiotic and/or thereactants and also with a higher yield of β-lactam antibiotic per amountof β-lactam nucleus employed and/or per amount of acylation agentemployed. This also results in a higher production capacity.

In addition, it is known from literature, for example WO-A-92/01061,that a high yield of β-lactam antibiotic per amount of β-lactam nucleuscan be obtained by applying a high molecular ratio between the acylationagent and the β-lactam nucleus. However, a drawback of applying a highmolecular ratio between the acylation agent and the β-lactam nucleus isthat large amounts of acylation agent are lost as a result of hydrolysisof the acylation agent (and possibly the β- lactam antibiotic).Consequently, a low synthesis/hydrolysis ratio (S/H), the molar ratiobetween synthesis product (β-lactam antibiotic) and hydrolysis product,is realized. Moreover, it has been found that working up the β-lactamantibiotic is often hampered by a relatively large amount of hydrolyzedacylation agent relative to β-lactam antibiotic being present in thereaction mixture obtained after the enzymatic acylation reaction, as aresult of which a smaller amount of β-lactam antibiotic can be isolated.

For the purposes of the present invention, the yield of β-lactamantibiotic per amount of reactant (β-lactam nucleus or acylation agent)to be achieved in the acylation reaction means the (molar) amount ofβ-lactam antibiotic formed in the acylation reaction per (molar) amountof reactant employed.

For the purposes of the present invention, the solubility of a compoundin a mixture means the dissolved concentration of the compound in thepresence of all other components of the mixture, expressed in mmol/litreor mass %. The solubility is measured by dissolving the compound atconstant pH and temperature and in the presence of all components ofmixture. Thereafter, the solubility can be calculated from the amount ofthe compound dissolved on reaching equilibrium (saturated solution).

For the purposes of the present invention, a compound is supersaturatedin a mixture when the dissolved concentration of that compound in themixture is greater than the solubility. The supersaturation factor meansthe ratio between the two aforementioned solubilities (supersaturateddivided by saturated). The supersaturation factor to be achieved and thetime during which supersaturation is maintained depend on a number offactors such as the nature and concentration of the compound, the natureand concentrations of the other components in the mixture, the pH andthe temperature. The supersaturation factor to be obtained dependslargely on the compound involved and is preferably larger than 2, moreparticularly larger than 5.

The concentration of the dissolved β-lactam nucleus is expressed as theamount of dissolved β-lactam nucleus in moles per kg of liquid reactionmixture; the total concentration of dissolved and undissolved β-lactamnucleus is expressed as the amount of β-lactam nucleus in moles per kgof the total reaction mixture; the total reaction mixture may contain,besides the solution, a plurality of solids, for example β-lactamnucleus, β-lactam antibiotic, (hydrolyzed) acylation agent andimmobilized enzyme. Similar definitions are applicable for the acylationagent and the β-lactam antibiotic.

A mixture in which the β-lactam nucleus or the acylation agent,respectively, is supersaturated can be obtained by means of for examplea pH shift. To in that end, if necessary, a concentrated mixture canfirst be prepared as a slurry or a solution by dissolving β-lactamnucleus or acylation agent, respectively, present in solid form with theaid of for example a pH increase or a pH decrease, or a pH decrease,respectively. It is preferred for the β-lactam nucleus and/or theacylation agent to be dissolved in the mixture obtained. However, it isalso possible for a portion of the β-lactam nucleus and/or the acylationagent to be still present in solid form. Subsequently, this slurry orsolution can be subjected to a pH decrease or a pH increase, or a pHincrease, respectively. In this way, a slurry or solution is obtained inwhich the β-lactam nucleus or acylation agent is supersaturated.

Any solid β-lactam nucleus present can be dissolved by for exampledecreasing the pH until a pH lower than 3, preferably lower than 2, inparticular lower than 1 is reached; or by increasing the pH to a pHhigher than 6, preferably higher than 7, in particular higher than 8. Inpractice, the final pH is preferably chosen such that the β-lactamnucleus goes only just completely into solution so that as concentrateda solution as possible is obtained. In practice, the concentratedsolution will usually have a concentration of the β-lactam nucleus of atleast 5 wt. %. The final pH will usually be lower than 10 and greaterthan 0.

Subsequently, a supersaturated solution can be obtained from a solution,whose pH may or may not have been decreased or increased, by increasingor decreasing the pH to a value between for example 3.0 and 9.0,preferably between 4.0 and 8.5, in particular between 4.5 and 8.0.

Any solid acylation agent present can be dissolved by for exampledecreasing the pH until a value lower than 8.0, preferably lower than6.5, in particular lower than 5.0 is reached; preferably, the final pHis chosen such that the acylation agent is only just completelydissolved and thus as concentrated a solution as possible is obtained.In practice, the concentrated solution will have a concentration of theacylating agent of at least 5 wt. %. The final pH will usually begreater than 1.

A mixture supersaturated with for example the acylation agent can beobtained from a mixture, preferably a solution, whose pH may optionallyhave been reduced, by increasing the pH to a value greater than forexample 4.5, preferably greater than 5.5, in particular greater than6.0.

Another manner of obtaining a mixture in which the β-lactam nucleusand/or the acylation agent are/is supersaturated is for example by atemperature decrease, optionally after any solid β-lactam nucleus and/orsolid acylation agent present have/has first at least partly beendissolved by a temperature increase or pH change.

Dissolution can be effected by for example a temperature increase untilfor example (virtually) all solid matter is in solution, for example toa temperature higher than 15° C., preferably higher than 20° C., inparticular higher than 25° C. Subsequently, a supersaturated slurry orsolution can be obtained from the obtained mixture by decreasing thetemperature to a temperature lower than 20° C., preferably lower than15° C., in particular lower than 10° C.

The supersaturated mixture preferably is prepared by changing the pHinasmuch as a higher supersaturation factor can then be achieved. Inpractice, a pH change and a temperature decrease will usually be appliedsimultaneously in the preparation of a supersaturated solution.

In a suitable embodiment of the process according to the invention firsta mixture in which the β-lactam nucleus and/or the acylation agentare/is supersaturated is prepared, whereupon the acylation reaction isstarted by for example adding (immobilized) enzyme. The concentration ofthe reactants will decrease during the acylation reaction so thatsupersaturation will have gone by the end of the acylation reaction. Inanother suitable embodiment the acylation reaction is started with aportion of the β-lactam nucleus and/or the acylation agent, which may ormay not be supersaturated, whereupon supersaturation is eithermaintained or brought about by adding to the reaction mixture, forexample by titration, a concentrated mixture of the β-lactam nucleusand/or the acylation agent with a different pH or a higher temperaturethan the pH or temperature at which the acylation reaction is carriedout. In practice, the β-lactam nucleus and the acylation agent may bothbe present in supersaturated condition during the acylation reaction,for example by metering into the acylation reactor a concentratedsolution or slurry of the β-lactam nucleus (with a high pH or optionallywith a low pH) and at the same time a concentrated solution or slurry ofthe acylation agent (with a low pH). In so doing, the pH can if desiredbe kept constant during the acylation reaction by for example titration.

The process according to the invention can very suitably be used in thepreparation of cefaclor.

Cefaclor exhibits poor stability at high pH (>6.5) whilst the solubilityof the corresponding β-lactam nucleus(7-amino-3-chloro-cef-3-em-4-carboxylic acid; 7-ACCA) is low at those pHvalues (about 6.0-6.5) at which degradation of cefaclor still isrelatively low.

As a result, the yield of cefaclor was low at both relatively high andrelatively low pH, so that a technically/commercially attractive processwas not possible. The yield of cefaclor per amount of 7-ACCA employedand per amount of acylation agent employed could admittedly be improvedby using for example naphthol as a complexing agent; the use of suchtoxic auxiliary materials in the preparation of antibiotics is, however,disadvantageous in that they need to be completely removed, whichentails additional process steps, with a strongly negative effect onprocess economics. Surprisingly, it has been found that the processaccording to the invention allows an exceptionally high supersaturationfactor (>10) to be achieved so that the acylation reaction can becarried out at a relatively low pH (6.0-6.5) whilst, yet, theconcentration of the β-lactam nucleus is sufficiently high. Thus, it isnow possible to prepare cefaclor in high yields through enzymaticacylation without using auxiliary materials so that atechnically/commercially attractive process can be realized.

Another application of the process according to the invention lies inthe preparation of for example ampicillin by acylation of6-aminopenicillanic acid (6-APA) with the aid of D-phenyl glycine amide(FGA). Since 6-APA and ampicillin degrade relatively quickly at highconcentration and at high pH it is important to have the acylationreaction proceed as rapidly as possible at as low a pH as possible. Byfor example increasing the pH of a mixture of 6-APA and FGA to a valuebetween 7.0 and 8.0 and then immediately lowering it to a pH between 6.0and 6.5 and then immediately initiating the enzymatic reaction it hasbeen found possible to achieve, in a short period of time, a high yieldof ampicillin per amount of 6-APA employed and per amount of acylationagent employed, with a relatively high concentration of 6-APA beingpresent in the solution only very briefly and with degradation ofampicillin being kept low at the relatively low pH value. At the sametime, hydrolysis of FGA to D-phenyl glycine (FG) is restricted.

The process according to the invention can also be applied withadvantage in the enzymatic preparation of cephalexin through acylationof 7-aminodesacetoxycephalosporanic acid (7-ADCA) with the aid ofD-phenyl glycine amide (FGA). The acylation reaction is usually carriedout at a relatively high pH value, for example between 7.5 and 8.5, andis attended by (unwanted) hydrolysis of FGA into D-phenyl glycine (FG).The reaction can be accelerated, and hydrolysis of the acylation agentcan be limited, by applying the process according to the invention, forexample by first raising the pH of a mixture of FGA and 7-ADCA to forexample a pH between 8.0 and 9.0 and then acidifying the mixture againto a pH between 6.5 and 8.5.

Another example of the process according to the invention is itsapplication in the enzymatic preparation of amoxicillin from 6-APA andD-p-hydroxyphenyl glycine methyl ester (FGHM) and in the preparation ofcefadroxil from 7-ADCA and FGHM. The solubility of FGHM is relativelylow and decreases with increasing pH, whereas the solubility of 6-APAand 7-ADCA is also relatively low and decreases with decreasing pH. Ithas been found that subjecting a mixture of for example 6-APA or 7-ADCAand FGHM to a pH decrease to a value at which FGHM is virtuallycompletely dissolved (for example a pH value between 5 and 6) and nextto a pH increase to a value between for example 6 and 7.5 enables asupersaturation of FGHM by a factor of 3-5 to be achieved. Thus, theacylation reaction can be carried out at a somewhat higher pH, with ahigher concentration of dissolved β-lactam nucleus, whilst theconcentration of the acylation agent is also relatively high.

Another embodiment is to dissolve 7-ADCA to a relatively highconcentration with a base to a pH between for example 8 and 9 and thenadding a concentrated, acid solution of FGHM, in which process the pHdecreases. In this embodiment, supersaturation of both 7-ADCA and FGHMcan be achieved at the same time. The enzymatic reaction can be startedafter all reactants have been added. It is also possible to add (aportion of the) concentrated FGHM or 7-ADCA solution (or slurry) duringthe reaction.

Another example of the process according to the invention is itsapplication in the enzymatic preparation of cefazolin fromtetrazole-1-acetic acid and as a β-lactam nucleus 7-aminocephalosporanicacid (7-ACA) or7-amino-3-(5-methyl-1,3,4-thiadiazole-2-yl-thiomethyl)-3-cef-em-4-carboxylicacid (7-ACA-MMTD). As the acylating agent does not contain an ax-aminogroup, a so-called thermodynamically controlled coupling reaction withthe acid may be performed. The optimum pH for such thermodynamicallycontrolled coupling reaction is preferably between 4.0 and 6.5. At thispH the solubility of the β-lactam nucleus is relatively low. It hasappeared that the conversion in the coupling reaction can be stronglyenlarged by adding a concentrated solution of the β-lactam nucleus witha relatively high pH (for instance between 7.0 and 9.0) to the enzymaticreaction mixture, while the pH of the reaction mixture is kept at avalue between 4.0 and 6.5, e.g. by titration with an acid.

The process according to the invention can suitably be applied in thepreparation of β-lactam antibiotics, for example cephalexin, ampicillin,cefaclor, amoxicillin, cephradine, cefadroxil, cefotaxime, cefazolin,cefprozil, loracarbef and cefaloglycin.

Any β-lactam nucleus can in principle be used, in particular a β-lactamnucleus with the general formula (1)

where R₀ represents H or an alkoxy group having 1-3 C atoms; Yrepresents CH₂, O, S or an oxidized form of sulphur; and Z represents

where R₁ represents for example H, OH, halogen, an alkoxy group having1-5 C atoms, an alkyl group having 1-5 C atoms, a cycloalkyl grouphaving 4-8 C atoms, an aryl or a heteroaryl group having 6-10 C atoms,in which the groups may or may not be substituted with for example analkyl, an aryl, a carboxy or an alkoxy group having 1-8 C atoms; andwhere the carboxylic acid group may be an ester group if so desired.

Suitable examples of β-lactam nuclei that may be employed in the processaccording to the invention are penicillin derivatives, for example6-aminopenicillanic acid (6-APA) and cephalosporic acid derivatives, forexample a 7-aminocephalosporanic acid with or without a substituent atthe 3-site, for example 7-aminocephalosporanic acid (7-ACA),7-aminodesacetoxycephalosporanic acid (7-ADCA),7-amino-3-chloro-cef-3-em-4-carboxylic acid (7-ACCA),7-amino-3-(1-propenyl)-cef-3-em-4-carboxylic acid (7-PACA),7-amino-3-(5-methyl-1,3,4-thiadiazole-2-yl-thiomethyl)cef-3-em-4-carboxylicacid (7-ACA-MMTD) and7-amino-3-chloro-8-oxo-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid.

In the (enzymatic) acylation reaction, the acylation agent may be forinstance a phenyl glycine in activated form, preferably a (primary,secondary or tertiary) amide or salt thereof, or a lower alkyl (1-4C)ester, for instance a methyl ester; suitable phenyl glycines are forexample substituted and unsubstituted phenyl glycines, in particularphenyl glycine, β-hydroxyphenyl glycine, dihydrophenyl glycine. Inaddition α-substituted acetic acid derivatives and the correspondingamides and esters may be applied, for instance phenyl acetic acid,phenoxy acetic acid, tetrazole-1-acetic acid, mandelic acid or thienylacetic acid.

In principle, any enzyme that is suitable as a catalyst in the couplingreaction can be used as the enzyme. Such enzymes include the enzymescollectively referred to as penicillin amidase or penicillin acylase.Such enzymes are described in for example J. G. Shewale et al., ProcessBiochemistry, August 1989, pp. 146-154 and in J. G. Shewale et all,Process Biochemistry International, June 1990, pp. 97-103. Examples ofsuitable enzymes are enzymes derived from Acetobacter, in particularAcetobacter pasteurianum, Aeromonas, Alcaligenes, in particularAlcaligenes faecalis, Aphanocladium, Bacillus sp., in particularBacillus megaterium, Cephalosnorium, Escherichia, in particularEscherichia coli, Flavobacterium, Fusarium, in particular Fusariumoxysporum and Fusarium solani, Kluyvera, Mycoplana, Protaminobacter,Proteus, in particular Proteus rettgari, Pseudomonas and Xanthomonas, inparticular Xanthomonas citrii.

Preferably an immobilized enzyme is used, since in that case the enzymecan be easily isolated and re-used.

Particularly suitable enzymes among the immobilized enzymes that arecommercially available are for example the Escherichia coli enzyme fromBoehringer Mannheim GmbH, which is commercially available under the nameEnzygel^(R), the immobilized Penicillin-G acylase from Recordati and theimmobilized Penicillin-G acylase from Pharma Biotechnology Hannover. Inaddition, enzymes may also be utilized in crystalline form (CLEC's™).

The temperature at which the enzymatic acylation reaction is effectedusually is below 40° C., preferably between −5 and 35° C. The pH atwhich the enzymatic acylation reaction is effected usually is between3.0 and 9.5, preferably between 4.0 and 9.0. The optimum pH for akinetically controlled coupling reaction is relatively high, forinstance between 4.5 and 9.0, preferably between 5.5 and 8.5, inparticular between 6.0 and 8.0. The optimum pH of a thermodynamicallycontrolled coupling reaction generally is lower and lies for instancebetween 3.0 and 7.0, preferably between 4.0 and 6.5.

The reaction preferably is stopped almost completely when maximumconversion has virtually been achieved. A suitable embodiment forstopping the reaction is to lower the pH, preferably to a value between4.0 and 6.3, in particular between 4.5 and 5.7. Another suitableembodiment is to lower the temperature of the reaction mixture onattaining the maximum conversion. A combination of the two embodimentsis possible also.

Once the reaction has been stopped on attaining the maximum conversion,the reaction mixture usually is present in the form of a suspensioncomprising a plurality of solids, for example the antibiotic, D-phenylglycine and, possibly, immobilized enzyme. The immobilized enzymepreferably is recovered in the interest of process economics. This cansuitably be accomplished by for example by filtering the reactionmixture on a sieve, while stirring, the stirrer's direction of rotationbeing chosen so that the suspension is pumped upwards at the centre ofthe stirrer. Subsequently, valuable components such as the antibioticand FG can be recovered by means of for example a pH change.

For the purposes of the invention, a pH change can be brought about byadding an acid. Suitable acids are for example mineral acids, inparticular sulphuric acid, hydrochloric acid or nitric acid andcarboxylic acids, for example acetic acid, oxalic acid and citric acid.A pH increase can be brought about by for example adding a base.Suitable bases are for example inorganic bases, in particular ammoniumhydroxide, potassium hydroxide or sodium hydroxide, and organic bases,for example triethyl amine and FGA. Preferably, ammonium hydroxide isused.

The enzymatic acylation reaction and the measures mentioned, for examplethe preparation of the supersaturated mixtures, can be effected inwater. If desired, the reaction mixture may also contain an organicsolvent or a mixture of organic solvents, preferably less than 30 vol.%. Examples of suitable organic solvents are alcohols having 1-7 Catoms, for example a monoalcohol, in particular methanol or ethanol; adiol, in particular ethylene glycol, or a triol, in particular glycerol.

The molar ratio of acylation agent to β-lactam nucleus, i.e. the totalamount of acylation agent added, divided by the total amount of β-lactamnucleus added, expressed in moles, is less than 2.5. It is preferred forthe molar ratio to be between 0.5 and 2.0, in particular between 0.7 and1.8.

The enzymatic acylation reaction is preferably carried out as a batchprocess. If desired, the reaction can also be carried out continuously.

The invention will be further elucidated by means of the followingexamples, without however being restricted thereto.

Abbreviations

7-ACCA: 7-amino-3-chloro-cef-3-em-4-carboxylic acid

7-ADCA: 7-aminodesacetoxycephalosporanic acid

6-APA: 6-amino-penicillanic acid

10 AMPI: ampicillin

CCl: cefaclor

CEX: cephalexin

FG: D-phenyl glycine

FGA: D-phenyl glycine amide

FGH: D-p-hydroxyphenyl glycine

FGHM: D-p-hydroxyphenyl glycine methyl ester

Assemblase™ is an immobilized Escherichia coli penicillin acylase fromE. coli ATCC 1105 as described in WO-A-97/04086. The immobilization iseffected as set out in EP-A-222462, with gelatin and chitosan being usedas gelating agents and glutaraldehyde as crosslinking agent.

The ultimate activity of the Escherichia coli penicillin acylase isdetermined by the amount of enzyme added to the activated globules andamounted to 3 ASU/g of dry weight, 1 ASU (Amoxicillin Synthesis Unit)being defined as the amount of enzyme capable of producing 1 g ofAmoxicillin.3H₂O from 6-APA and FGHM per hour (at 20° C.; 6.5% 6-APA and6.5% FGHM).

Comparative Example 1 Synthesis of Cefaclor (7-ACCA not Supersaturated)

An enzyme reactor (1.5 l, diameter 11 cm), fitted with a 175 μm meshsieve bottom, was filled with 100 g of net-wet Assemblase™.

A preparation reactor (1.2 l) was filled with 75.4 g of FGA (0.500mole), 4.0 g of sodium bisulphite, 700 g of water, 6.6 g of 4N H₂SO₄ and72.1 g of 7-ACCA (0.300 mol). This mixture was stirred at T=10° C.; thepH was 7.5. Subsequently, the mixture was transferred into the enzymereactor with the aid of 118 ml of water (T=10° C.).

The stirrer in the enzyme reactor was switched on at t=0. Thetemperature was kept at 10° C. The pH was kept at 7.5 by titration with4N H₂SO₄. At t=48 minutes the enzyme reactor contained approx.:

215 mmol CCl (conversion=72%)

70 mmol 7-ACCA

175 mmol FGA

95 mmol FG

At t=81 minutes the enzyme reactor contained approx.:

212 mmol CCl (conversion=71%)

61 mmol 7-ACCA

83 mmol FGA

182 mmol FG

Thereafter, the amount of CCl decreased.

EXAMPLE I Synthesis of Cefaclor (7-ACCA Supersaturated)

An enzyme reactor (1.5 l, diameter 11 cm), fitted with a 175 μm meshsieve bottom, was filled with 300 g of net-wet Assemblase™.

A preparation reactor (1.2 l) was filled with 86.6 g of 7-ACCA (0.360mol), 67.8 g of FGA (0.450 mol), 4.0 g of sodium bisulphite and 402 g ofwater. This mixture was stirred for 5 minutes at T=10° C.; the pH was7.4.

The pH was brought to 8.0, while stirring, with the aid of 16.8 g ofconcentrated ammonium hydroxide. Subsequently, the pH was lowered from8.0 to 6.4 by metering in 73.8 ml of 4N H₂SO₄ in 20 minutes. Next, att=0, the mixture was transferred from the preparation reactor to theenzyme reactor with the aid of 140 ml of water (T=10° C.)

The stirrer in the enzyme reactor was switched on at t=0; T=10° C. ThepH was kept at 6.4 by titration with 4N H2SO₄. After 7 hours, 66.0 ml ofacid had been added. The reactor now contained:

300 mmol CCl (conversion=83%)

55 mmol 7-ACCA

100 mmol FGA

45 mmol FG

EXAMPLE II Synthesis of Cefaclor (7-ACCA Supersaturated and 7-ACCA BeingMetered in During the Enzymatic Reaction)

An enzyme reactor (1.5 l, diameter 11 cm), fitted with a 175 μm meshsieve bottom, was filled with 150 g of net-wet Assemblase™.

A preparation reactor (1.2 l) was filled with 48.1 g of 7-ACCA (0.200mol), 75.4 g of FGA (0.500 mol), 4.0 g of sodium bisulphite and 175 g ofwater. This mixture was stirred for 5 minutes at T=10° C.; the pH was7.43.

The pH was brought to 9.0, while stirring, with the aid of 15.5 g ofconcentrated ammonium hydroxide. Subsequently, the pH was lowered from9.0 to 6.4 by metering in 80.6 ml of 6N H₂SO₄ in 45 minutes. Next, att=0, the mixture was transferred from the preparation reactor to theenzyme reactor with the aid of 40 g of water (T=10° C.).

The stirrer in the enzyme reactor was switched on at t=0. Thetemperature was kept at T=10° C. 244 g (0.200 mol) of 7-ACCA solutionwere metered in at a constant rate in 109 minutes. The solution had beenfreshly prepared by suspending 48.1 g of 7-ACCA (0.200 mol) in 183 g ofwater at T=3° C. and raising the pH to 8.2 with the aid of 13.2concentrated NH₃, in which process all 7-ACCA dissolved. From t=0onwards, the pH in the enzyme reactor was kept at 6.4 by titration with6N H₂SO₄. At t=350 minutes the enzyme reactor contained:

350 mmol CCl (conversion=88%)

45 mmol 7-ACCA

80 mmol FGA

65 mmol FG

At t=400 minutes the amount of Cefaclor was maximum and the pH waslowered to 5.0 by adding 6N H₂SO₄. The enzyme reactor now contained:

370 mmol CCl (conversion=92%)

25 mmol 7-ACCA

40 mmol FGA

85 mmol FG

Comparative Experiment B Synthesis of Ampicillin (6-APA notSupersaturated)

First a solution of FGA. 1/2 H₂SO₄ was prepared. 301.6 g of FGA (2.00mol) were suspended in 650 g of water at T=5° C. 102.1 g of 96% H₂SO₄(1.00 mol) were added dropwise while stirring, with the temperaturebeing kept at T<25° C. by means of cooling.

Next the enzymatic condensation was performed. An enzyme reactor (1.5 l,diameter 11 cm), fitted with a 175 μm mesh sieve bottom, was filled with300 g of net-wet Assemblase™.

A preparation reactor (1.2 l) was filled with 131.6 g of 6-APA (0.600mol), 30.2 g of FGA (0.200 mol) and 400 ml of water (T=10° C.). Thismixture was stirred for 15 minutes at T=10° C. and subsequently, at t=0transferred into the enzyme reactor with the aid of 100 ml of water(T=10° C.)

The stirrer in the enzyme reactor was switched on at t=0. 423.7 g (0.800mol) of FGA. 1/2H₂SO₄ were added to the solution at a constant rate in283 minutes, with the temperature being kept at 10° C. The pH was 6.3From t=328 minutes onwards, the pH was kept at 6.3 by titration with 6NH₂SO₄.

At t=540 minutes the amount of Ampicillin was maximum and the pH waslowered to 5.6 by adding 6N H₂SO₄.

The enzyme reactor now contained:

575 mmol AMPI (=96% relative to the amount of 6-APA employed)

15 mmol 6-APA

50 mmol FGA

365 mmol FG

EXAMPLE III Synthesis of Ampicillin (6-APA Supersaturated)

An enzyme reactor (1.5 l, diameter 11 cm), fitted with a 175 μm meshsieve bottom, was filled with 300 g of net-wet Assemblase™.

A preparation reactor (1.2 l) was filled with 550 ml of water (T=10°C.), 138.8 g (0.920 mol) of FGA and 131.6 g (0.600 mol) of 6-APA. After5 minutes at 10° C. a clear solution obtained with a pH value of 7.4.Subsequently, the pH was brought to 6.5 with 96% H₂SO₄ (ca. 16 g) andafter stirring for 5 minutes at 10° C. the solution was transferred tothe enzyme reactor at t=0 with the aid 100 ml of water (T=10° C.).

At t=0 the temperature was 10° C. and the pH =6.3. During the enzymereaction the pH was kept at 6.3 by titration with 6N H₂SO₄. At t=300minutes the amount of AMPI was maximum and the pH was lowered to 5.6 byadding 6N H₂SO₄.

The enzyme reactor now contained:

575 mmol AMPI (=96% relative to the amount of 6-APA employed)

15 mmol of 6-APA

40 mmol of FGA

295 mmol PG

Comparative Example C Synthesis of Cephalexin (7-ADCA notSupersaturated)

An enzyme reactor (1.5 l, diameter 11 cm), fitted with a 175 μm meshsieve bottom, was filled with 75 g of net-wet Assemblase™.

A preparation reactor (1.2 l) was filled with 668 g of water (T=4° C.),4.0 g of sodium bisulphite, 130.3 g of 7-ADCA (0.600 mol) and 75.4 g ofFGA (0.500 mol). 7.8 g of concentrated ammonium hydroxide were added,whereupon the suspension was stirred for 15 minutes at T=4° C. The pHwas 7.8.

Subsequently, at t=0, the suspension was transferred into the enzymereactor with the aid of 50 ml of water (T=4° C.). The stirrer in theenzyme reactor was switched on at t=0. The temperature was kept at T=4°C. all the time. All 7-ADCA had dissolved after approximately 75minutes; thereafter, a clear solution, apart from solid Assemblase™, waspresent. After 210 minutes the pH had risen to 8.6.

The reactor now contained:

393 mmol CEX (conversion=66%; S/H=6.1)

200 mmol 7-ADCA

64 mmol FG

33 mmol FGA.

EXAMPLE IV Synthesis of Cephalexin (7-ADCA Supersaturated)

An enzyme reactor (1.5 l, diameter 11 cm), fitted with a 175 μm meshsieve bottom, was filled with 75 g of net-wet Assemblase™.

A preparation reactor (1.2 l) was filled with 600 ml of water (T=4° C.),4.0 g of sodium bisulphite, 130.3 g of 7-ADCA (0.600 mol) and 90.5 g ofFGA (0.600 mol). The suspension was stirred for 15 minutes at T=4° C.The pH was 7.6.

The pH was brought to 8.6, with stirring, using 43.8 g of concentratedammonium hydroxide. Stirring was effected for 5 minutes at T=4° C.Subsequently, 21.2 g of concentrated H₂SO₄ were added. The clearsolution was stirred for 15 minutes at T=4° C. The pH was 7.4.

Subsequently, at t=0, the suspension was transferred into the enzymereactor with the aid of 50 ml of water (T=4° C.). The stirrer in theenzyme reactor as switched on at t=0. The temperature was kept at T=4°C. all the time. At t=90 minutes the pH had risen to 8.05. 5.5 g ofconcentrated H₂SO₄ were added, causing the pH to decrease to 7.75. Aclear solution, apart from solid Assemblase™, was present throughout theenzyme reaction.

After 340 minutes the pH had risen to 8.5.

The reactor now contained:

445 mmol CEX (conversion=74%; S/H=7.2)

146 mmol 7-ADCA

62 mmol FG

80 mmol FGA.

EXAMPLE V Synthesis of Cephalexin (7-ADCA Supersaturated)

An enzyme reactor (1.5 l, diameter 11 cm), fitted with a 175 μm meshsieve bottom, was filled with 75 g of net-wet Assemblase™.

A preparation reactor (1.2 l) was filled with 500 ml of water (T=4° C.), 4.0 g of sodium bisulphite, 162.9 g of 7-ADCA (0.750 mol) and 113.1 gof FGA (0.750 mol). The suspension was stirred for 15 minutes at T=4° C.The pH was 7.6.

The pH was brought to 8.7, with stirring, using 55.6 g of concentratedammonium hydroxide. Stirring was effected for 5 minutes at T=4° C.Subsequently, 9.6 g of concentrated H₂SO₄ were added. The clear solutionwas stirred for 15 minutes at T=4° C. The pH was 8.0.

Subsequently, at t=0, the suspension was transferred into the enzymereactor with the aid of 50 ml of water (T=4° C.). The stirrer in theenzyme reactor was switched on at t=0. The temperature was kept at T=4°C. all the time. After 60 minutes the pH had risen to 8.3. The pH wasnow kept at 8.3 by titration with concentrated H₂SO₄.

At t=300 minutes a total of 23.4 g of concentrated H₂SO₄ had beentitrated. At this point titration was stopped; at t=450 minutes the pHhad risen to 8.7.

A clear solution, apart from solid Assemblase™, was present throughoutthe enzyme reaction.

At t=450 minutes the reactor contained:

545 mmol CEX (conversion=73%; S/H=8.5)

195 mmol 7-ADCA

64 mmol FG

129 mmol FGA.

Comparative Experiment D Synthesis of Cefadroxil (neither 7-ADCA norFGHM Supersaturated)

An enzyme reactor (1.5 l, diameter 11 cm), fitted with a 175 μm meshsieve bottom, was filled with 300 g of net-wet Assemblase™.

A preparation reactor (1.2 l) was filled with 63.7 ml of 7-ADCA (0.293mol), 107.1 g of FGHM (0.590 mol), 4.0 g of sodium bisulphite and 440 gof water. This mixture was stirred for 5 minutes at T=10° C. with the pHbeing kept at 7.0 with the aid of 7.5 g of concentrated ammoniumhydroxide.

At t=0, the suspension was transferred from the preparation reactor intothe enzyme reactor with the aid of 50 ml of water (T=10° C.). Thestirrer in the enzyme reactor was switched on at t=0. The temperaturewas kept at T=10° C.; the pH remained constant at 7.0 (no titration).

After 380 minutes the enzyme reactor contained:

68 mmol Cefadroxil (conversion=23%; S/H=0.67)

222 mmol 7-ADCA

420 mmol FGHM

101 mmol FGH

EXAMPLE VI Synthesis of Cefadroxil (both FGHM and 7-ADCA Supersaturated)

An enzyme reactor (1.5 l, diameter 11 cm), fitted with a 175 μm meshsieve bottom, was filled with 270 g of net-wet Assemblase™.

A preparation reactor (1.2 l) was filled with 97.7 ml of 7-ADCA (0.448mol), 4.0 g of sodium bisulphite and 250 g of water (T=10° C.). Thesuspension was stirred for 5 minutes at T=10° C. Subsequently, the pHwas brought to 8.1 with the aid of 40.3 g of NH₃, in which process aclear solution evolved (T=10° C.).

A second preparation reactor was filled with 98.5 g of FGHM (0.538 mol),60 g of water (T=10° C.) and 105.1 g of 6N H₂SO₄ solution. This solutionwas brought to T=10° C.; pH=2.3.

The 7-ADCA solution from the first preparation reactor was transferredto the enzyme reactor with the aid of 20 ml of water (T=10° C.). Thestirrer in the enzyme reactor was switched on. From t=0 the FGHMsolution from the second preparation reactor was metered into the enzymereactor at a constant rate in 120 minutes. The temperature was kept atT=10° C.

After 30 minutes the pH had decreased from 8.1 to 6.8. Next, the pH wasmaintained at 6.8 by titration with a concentrated ammonium hydroxide.At t=120 minutes, 8.3 g of concentrated ammonium hydroxide had beenadded. Titration was stopped; the pH continued to be 6.8.

After 420 minutes the enzyme reactor contained:

390 mmol Cefadroxil (conversion 87%; S/H=4.0

50 mmol 7-ADCA

44 mmol FGHM

97 mmol FGH

What is claimed is:
 1. Process for the preparation of a β-lactamantibiotic in which a β-lactam nucleus is subjected to an enzymaticacylation reaction with the aid of an acylation agent at a molar ratioof acylation agent/β-lactam nucleus of less than 2.5, wherein theacylation agent and/or the β-lactam nucleus are/is supersaturated in thereaction mixture during at least part of the acylation reaction. 2.Process according to claim 1, in which the acylation agent and/or theβ-lactam nucleus are/is supersaturated in the reaction mixture at thebeginning of the acylation reaction.
 3. Process according to claim 1, inwhich a concentrated slurry or solution of the β-lactam nucleus and/orthe acylation agent with a different pH or a higher temperature than thepH or temperature at which the acylation reaction is carried out isadded to the reaction mixture during the acylation reaction.
 4. Processaccording to claim 1 in which the β-lactam nucleus is supersaturated inthe reaction mixture.
 5. Process according to claim 4 in which a mixturein which the β-lactam nucleus is dissolved is subjected to a pH decreaseor a pH increase until a pH between 3.0 and 9.0 is reached.
 6. Processaccording to claim 5 in which the pH decrease or pH increase is effecteduntil a pH between 4.0 and 8.5 is reached.
 7. Process according to claim1 in which the acylation agent is supersaturated in the reactionmixture.
 8. Process according to claim 7 in which a mixture in which theacylation agent is dissolved is subjected to a pH increase.
 9. Processaccording to claim 8 in which the pH is increased to a pH higher than5.5.
 10. Process according to claim 9 in which the pH is increased to apH higher than
 6. 11. Process according to claim 4 in which a mixturecontaining dissolved β-lactam nucleus and/or acylation agent issubjected to a temperature decrease.
 12. Process according to claim 11in which the temperature is decreased to a temperature below 15° C. 13.Process according to claim 12 in which the temperature is decreased to atemperature below 10° C.
 14. Process according to claim 7 in which themethyl ester of p-hydroxyphenyl glycine is used as acylation agent. 15.Process according to claim 1 in which an amide is used as acylationagent.
 16. Process according to claim 1 in which7-aminodesacetoxycephalosporanic acid (7-ADCA),7-amino-3-chloro-cef-3-em-4-carboxylic acid (7-ACCA),6-aminopenicillanic acid (6-APA), 7-aminocefalosporanic acid (7-ACA),7-amino-3-(1-propenyl)-cef-3-em-4-carboxylic acid (7-PACA),7-amino-3-(5-methyl-1,3,4-thiadiazole-2-yl-thiomethyl)-cef-3-em-4-carboxylicacid (7-ACA-MMTD) or7-amino-3-chloro-8-oxo-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid isused as β-lactam nucleus.